Pulmonary arterial hypertension (PAH) is a severe disease characterized by excessive proliferation of apoptosis-resistant pulmonary artery endothelial cells (PAEC) and smooth muscle cells (PASMC), progressive pulmonary arterial (PA) stiffening, and ultimately right heart failure and death. Recent studies suggest that increased PA stiffness contributes significantly to increased right ventricular afterload and is associated with increased mortality in PAH patients, however the role of PA stiffening in the pathogenesis of PAH has not yet been fully elucidated. We have used atomic force microscopy (AFM) microindentation to mechanically characterize the stiffness of pulmonary arteries at an unprecedented micro-scale level in experimental PAH. Our preliminary findings demonstrate that distal pulmonary arteries develop significant increases in matrix stiffness by more than three-fold in the rat models of SU5416/hypoxia and monocrotaline (MCT)-induced PAH. Furthermore, human PASMC and PAEC grown on polyacrylamide substrates with the stiffness of remodeled pulmonary arteries develop a striking hyperproliferative phenotype, decreased expression of cyclooxygenase (COX)-2, reduced prostaglandin I2 synthesis, and increased secretion of endothelin-1. Taken together, our findings suggest that matrix remodeling in the PA wall fundamentally biases cellular behavior towards progressive vascular remodeling via previously unrecognized effects of matrix stiffening. We hypothesize that increases in PA stiffness are not merely a consequence of pathological alterations in the vessel wall, but rather that increases in matrix stiffness trigger a remodeling phenotype characterized by enhanced cellular proliferation and matrix deposition in pulmonary arteries, promoting mechanobiological feedback amplification of vascular remodeling. To test our hypothesis, we propose two specific aims. In Specific Aim 1, we will investigate the magnitude and distribution of pathological increases in pulmonary arterial stiffness at the micron spatial scale in human PAH tissue. We will use AFM microindentation to characterize the mechanical environment of remodeled pulmonary arteries in lung tissue derived from subjects with WHO Group I PAH and secondary pulmonary hypertension compared with normal vessels in lung tissue from control subjects. In Specific Aim 2, we will elucidate the mechanisms by which the mechanical environment promotes pathologic remodeling behaviors in PASMC and PAEC derived from subjects with PAH. We will investigate whether matrix stiffness regulates the biology of proximal and distal PASMC and PAEC derived from subjects with PAH compared with control subjects. We will also determine whether stiffness-dependent attenuation of COX-2- derived prostanoid biosynthesis drives progressive vascular remodeling in a mechanobiological feedback loop. The proposed studies will provide novel insights into the role of the mechanical environment in pulmonary vascular remodeling in human PAH and will elucidate the mechanisms activating the stiffness-dependent switch to a remodeling cellular phenotype in human PASMC and PAEC.